Optimization of a fragment size analysis tool for identification of Cryptosporidium species and Gp60 alleles infecting domestic ruminants

Abstract

A capillary electrophoresis (CE)-based DNA fragment analysis tool was optimized to identify in a single capillary the most common Cryptosporidium species and Cryptosporidium parvum GP60 alleles infecting domestic ruminants. For this purpose, a panel of genomic DNA samples including six Cryptosporidium species (C. parvum, C. bovis, C. ryanae, C. andersoni, C. ubiquitum, and C. hominis) and 18 C. parvum GP60 subtypes belonging to the subtype families IIa and IId was used. All these samples had been characterized previously by sequencing of SSU rRNA and GP60 genes. Isolates were re-amplified by PCR at these loci using sets of newly designed primers and subjected to CE. Fragment sizes were adjusted after comparison with sizes obtained by sequence analysis. The optimized CE-based approach provided fragments of different size for most Cryptosporidium species, but did not differentiate C. bovis and C. ryanae. Many of the GP60 subtypes (11/18) were also readily differentiated by CE, although overlapping in fragment sizes between IIa and IId subtypes was noticed. The CE-based tool was subsequently used to analyze Cryptosporidium isolates from naturally infected calves (n: 123) and lambs (n: 113) from farms in northern Spain. All isolates provided fragments typical of C. parvum. Fragment analysis at the GP60 locus differentiated a total of 10 alleles within isolates from calves (6 alleles) and lambs (8 alleles), with all but three alleles being host-associated. These findings support the validity of the optimized CE approach as a discriminatory and time- and cost-saving alternative to sequencing for identification of Cryptosporidium species and GP60 alleles in domestic ruminants.